Dietzia bacterium for treatment of disease

ABSTRACT

This invention relates to compositions comprising a bacterium of the genus  Dietzia  that is useful for treating paratuberulosis in ruminants and to a method for culturing the bacterium. The invention further relates to methods of treating Johne&#39;s disease by administering to a mammal a composition of the invention.

STATEMENT OF RELATED REFERENCES

This application claims priority to international patent applicationPCT/US03/17540, which claims priority to U.S. Provisional ApplicationNo. 60/385,232, filed Jun. 3, 2002, the entire disclosure of which isincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a novel bacterium and the use of thenovel bacterium and other members of its genus, active portions thereofand/or proteins therefrom to treat a variety of diseases. Morespecifically, the invention relates to the use of a bacterium specieswithin the genus Dietzia, including the bacterium Dietzia sp. C79793-74,and other bacteria isolated by an in vitro inhibition procedure used toisolate Dietzia, portions and/or proteins produced therefrom, to treatparatuberculosis of ruminants and mycobacterial-caused diseases ofhumans, such as Crohn's disease, leprosy, Sarcoidosis, and tuberculosis(ICE).

BACKGROUND OF TEE INVENTION

Paratuberculosis, or Johne's disease, in ruminants is a chronicinfection of the gastrointestinal tract. Mycobacterium paratuberculosis(MpT) is known to be the causative agent of Johne's disease. Mostanimals that contract Johne's disease become infected shortly afterbirth and do not become clinically ill until they are adults. Infectionis permanent, there are no viable treatments, and, if not culled, mostclinically sick animals eventually die of wasting.

Current estimates indicate that 3.4% of dairy cows in 21.6% of herds and0.9% of beef cattle in 7.9% of herds in the United States are infectedwith MpT. Similar data are reported in essentially all other countries.These results demonstrate that Johne's disease is a very seriousworldwide problem. Indeed, it has been estimated to have a $0.2-1.5billion dollar economic impact in the United States due to loss of milkincome, increased culling, low culling weights, extended calvingintervals, and the unmarketability of breeding stock. Similarly, largeeconomic losses from Johne's disease are also suffered in the sheepindustry.

It is generally accepted that for clinical manifestation ofparatuberculosis to occur, infection with MpT must occur at a young age.To date, no effective treatment has been reported for infected animals.Moreover, no prophylaxis has been reported that prevents infection.Biosecurity and other management practices have been proposed as a meansto control the spread of Johne's disease. It is agreed that the spreadof MpT could be lessened by: a) persistent attention to detail; b)avoidance of contact with fecal material from infected animals; and c)culling all infected and all offspring born to infected cattle. However,from a practical perspective, it has been suggested that managementalone will likely fail to control infections. Thus, there is a need forviable methods to prevent and control paratuberculosis.

Mycobacteria are major pathogens of humans, as well as animals. Thereare approximately ten million cases of tuberculosis worldwide with anannual mortality of three million. Leprosy, caused by Mycobacteriumleprae, afflicts over ten million people, primarily in developingcountries. Mycobacterium tuberculosis and mycobacteria of theMycobacterium avium-intracellulare group are major opportunisticpathogens of immuno-compromised patients such as AIDS patients. Crohn'sdisease and Sarcoidosis are postulated to be a result of MpT infection.Most treatments for these diseases require intense and lengthycombinational drug therapy. Such treatments not only allow resistantstrains to arise, but for Crohn's patients lasting resolution of diseasehas yet to be established. Thus, there is a need for more effectivetreatments for mycobacterial-incited diseases of humans.

Crohn's disease is a chronic, debilitating and potentially fatal diseasethat bears extensive clinical, pathologic, and systemic similarity toJohne's disease. In the United States, the number of newly diagnosedCrohn's patients is estimated to be 20,000 each year. Crohn's disease isa granulomatous ileo-colitis of unknown etiology. Postulates on theimmunopathogenesis of Crohn's disease are that the disease results froman antigenic challenge to the gut-associated lymphoid tissues (GALT).Once triggered, cytokines and other inflammatory mediators releasedresult in chronic and persistent inflammation. This inflammatoryreaction is postulated to be the result of hyper-responsiveness of GALTto antigens present within intestinal cells. This hyper-reactivity maybe a result of an immunoregulatory defect or from a persistent stimulus,such as MpT antigens. Recent evidence defining a genetic predispositionis consistent with this theory. Thus, there is a need for treatments forCrohn's disease that target MpT in particular.

Animals afflicted with Johne's disease may be the source of MpT thatunderlie Crohn's disease in humans. For example, MpT may be transmittedto humans through contaminated meat and/or pasteurized milk. MpT is anintracellular pathogen that colonizes and multiplies in phagocytic cellspresent in blood and other tissues. Since phagocytes are naturalconstituents of milk, it is not surprising that MpT is found in milk ofinfected cows. Moreover, MpT is partially resistant to pasteurizationpresently used commercially. Furthermore, animals that are heavilyinfected with MpT are usually culled and likely used in the productionof ground beef. Inadequate cooking would again result in live MpT beingpresent in food. Thus, any effective treatment of paratuberculosis islikely to reduce the transmission of MpT to humans, which could in turnresult in a lower incidence of Crohn's disease.

SUMMARY OF THE INVENTION

The present invention relates to the use of bacteria of the genusDietzia for the treatment of various diseases. The invention includesthe use of an isolated bacterium identified as Dietzia sp. C79793-74 anddeposited with the American Type Culture Collection (ATCC), located at10801 University Blvd, Manassas, Virgina 20110-2209 USA. as AccessionNumber PTA-4125 on Mar. 7, 2002, for the treatment of various diseases.

In one embodiment, the invention includes a composition suitable foradministration to an animal comprising a carrier and one or morebacteria, the bacteria species selected from the genus Dietzia,including ATCC Accession Number PTA4125, referred to collectively hereinas “The Bacteria”.

The invention further includes a composition suitable for administrationto an animal comprising a carrier and a pharmacologically active, orrecombinant DNA-derived, dose of The Bacteria, active fragments of TheBacteria, including active fragments of enzymes, active moleculessecreted from The Bacteria or from organisms with The Bacteria-derivedDNA, and combinations of the above. Active molecules include proteins,DNA molecules, RNA molecules, carbohydrates, and components of the cellenvelope, such as lipoglycans.

In another embodiment, the invention includes compositions that aresuitable for administering The Bacteria to animals affected by Johne'sdisease. For example, the invention encompasses feed compositions andfeed additives in which The Bacteria is an ingredient.

In still another embodiment, the invention includes a compositionsuitable for administration to a human comprising a suitable carrier andThe Bacteria.

The invention further includes a composition suitable for administrationto a human comprising a carrier and a pharmacologically active, orrecombinant DNA-derived, dose of The Bacteria, active fragments of TheBacteria, active molecules secreted from The Bacteria or from organismswith The Bacteria-derived DNA, and combinations of the above. Methodsfor producing bacteria through recombinant processes are well known inthe art.

In addition, the invention relates to methods of treating Johne'sdisease, Crohn's disease, and other mycobacterial-caused diseases byadministering a composition comprising The Bacteria. In one embodiment,the invention includes a method of treating Johne's disease comprisingorally administering a composition comprising The Bacteria to anymammals infected with MpT.

The bacteria used in the present invention are isolated from an animalsample, which may be the feces of bovine, co-culturing the bacteriumwith MpT, and selecting bacteria that inhibit the growth of MpT. Inother embodiments, the bacterium are (or bacteria is) isolated fromsoil, sea water, or even insects.

The method also relates to a method of culturing bacteria of the genusDietzia in a medium containing Tryptose soy broth.

DETAILED DESCRIPTION OF THE INVENTION

All ranges recited herein include all combinations and sub-combinationsincluded within that range; therefore, a range from “about 15 to about60” would include ranges from about 15 to about 45, from about 30 toabout 47, etc. A range of “up to 85” would include up to 80, up to 50,up to 24, etc.

The present invention relates to novel bacteria such as those specieswithin the Dietzia genus (“The Bacteria”), that, when co-cultured with apathogenic mycobacterium, inhibit the growth of the co-culturedmycobacterium and therefore prove useful in controlling the growth ofsaid mycobacterium.

The Bacteria have also surprisingly and unexpectedly proved to beeffective in treating MpT infected cattle, including those with andthose without clinical disease.

Specifically, administering The Bacteria to ruminants has solved alongstanding problem in that it has proved to be effective for treatingMpT-infected ruminants. It has been surprisingly and unexpectedlydiscovered that the oral administration of The Bacteria in apharmacologically active dose to cattle treats the symptoms of Johne'sdisease and results in long term health benefits to the administeredruminant.

The bacterium is effective to treat Johne's disease in any ruminant,including, but not limited to, bovine, ovine, caprine, and cervine.Administration of The Bacteria to clinically, asymptomatic MpT-infectedruminants is also useful in preventing the onset of clinical symptoms ofJohne's disease. In one embodiment, the ruminants include bovines, suchas dairy calves and adult lactating and nonlactating cows. In anotherembodiment, the ruminants include caprine or ovine.

The Bacteria in one embodiment is administered to the ruminant orally.The Bacteria may be administered in a solution, mixed with a feed and/ormixed with a feed additive. The oral administration may be given in aseries of doses administered daily for a period of at least 3 months.When the ruminant is a bovine, the treatment may be administered for atleast 6 months at a dosage of at least 10¹¹ cfu. This includes methodsof treatment where The Bacteria is administered at a dosage of at least10¹² cfu and further includes methods of treatment where The Bacteria isadministered at a dosage of at least 10¹³ cfu. In one embodiment, TheBacteria are administered orally to adult clinically-ill or asymptomaticdairy cows on a daily basis for about 3-10 months. However, anyacceptable time period may be used as long as a pharmacologicallyeffective dose is administered such that paratuberculosis is effectivelyprevented and/or cured. For example, if The Bacteria is mixed with afeed and/or feed additive, ruminants may be fed on a constant diet offeed containing a pharmacologically effective dose of The Bacteria untila total pharmacological dose results in cure and/or prevention ofdisease.

In one embodiment, 2×10¹¹ cfu of The Bacteria are administered daily, ina single dose. If The Bacteria are incorporated into feed, a range ofconcentrations of The Bacteria in the feed are appropriate to practicethe method of the invention, in one embodiment about 10¹¹-10¹³ cfu perday. In one embodiment, The Bacteria are incorporated into the feed at aconcentration such that 2×10¹¹ cfu of The Bacteria are administered inan amount of feed that is consumed in a single day. Any appropriateamount of The Bacteria, or active portions or proteins thereof, may beincorporated into the feed as long as a pharmacologically active dose ofthe material is fed to the animal within a daily feed. If The Bacteriaare incorporated into a feed additive, a range of concentrations of TheBacteria in the feed additive are appropriate to practice the method ofthe invention, in one embodiment about 10¹¹ cfu per day. In oneembodiment, The Bacteria are incorporated into the feed additive at aconcentration such that 2×10¹¹ cfu of The Bacteria are administered inan amount of feed that is consumed in a single day.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. The Bacteria can beprepared in any type of appropriate medium for administration. Forexample, The Bacteria can be provided together with physiologicallytolerable liquid (e.g., saline), gel or solid carriers, diluents,adjuvant, excipients and as a rumen-protected encapsulated ingredient.Suitable diluents and excipients include pharmaceutical grades ofphysiological saline, dextrose, glycerol, mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, magnesium carbonate,and the like, and combinations thereof. In addition, if desired thecompositions may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, stabilizing or pH buffering agents. Thesecompositions typically contain 1%-95% of active ingredient, preferably2%-70%. The Bacteria can be incorporated into tablets, boluses, orcapsules, and dosed to the patient. The Bacteria may also beincorporated into salt blocks and the like. The Bacteria can be added tofeed as a freeze-dried powder or as an encapsulated bacteria that isprotected from degradation in the rumen. In one embodiment, The Bacteriais prepared in a solution of physiological phosphate-buffered saline(PBS) or in the culture medium, Tryptose soybean broth (TSB) for oraladministration to a ruminant.

The Bacteria can be incorporated into tablets, drenches, boluses,capsules or premixes. Formulation of these active ingredients into suchdosage forms can be accomplished by means of methods well known in thepharmaceutical formulation arts. See, for example, U.S. Pat. No.4,394,377. (This patent, and all other references cited herein arehereby incorporated by reference.) Filling gelatin capsules with anydesired form of the active ingredients readily produces capsules. Ifdesired these materials can be diluted with an inert powdered diluent,such as sugar, starch, powdered milk, purified crystalline cellulose, orthe like to increase the volume for convenience of filling capsules.

Conventional formulation processes can be used to prepare tabletscontaining The Bacteria. In addition to the active ingredients, tabletsmay contain a base, a disintegrator, an absorbent, a binder, and alubricant. Typical bases include lactose, sugar, sodium chloride, starchand mannitol. Starch is also a good disintegrator as is alginic acid.Surface-active agents such as sodium lauryl sulfate and dioctyl sodiumsulphosuccinate are also sometimes used. Commonly used absorbentsinclude starch and lactose. Magnesium carbonate is also useful for oilysubstances. As a binder there may be used, for example, gelatin, gums,starch, dextrin, polyvinyl pyrrolidone and various cellulosederivatives. Among the commonly used lubricants are magnesium stearate,talc, paraffin wax, various metallic soaps, and polyethylene glycol.

Drenches are prepared most readily by choosing a saline-suspended formof The Bacteria, fragments thereof or active molecules secretedtherefrom. A water-soluble form of one ingredient may be used inconjunction with a water-insoluble form of the other by preparing asuspension of one with an aqueous solution of the other. Water-insolubleforms of either active ingredient may be prepared as a suspension or insome physiologically acceptable solvent such as polyethylene glycol.Suspensions of water-insoluble forms of either active ingredient can beprepared in oils such as peanut, corn, sesame oil or the like; in aglycol such as propylene glycol or a polyethylene glycol; or in waterdepending on the solubility of a particular active ingredient. Suitablephysiologically acceptable adjuvants may be necessary in order to keepthe active ingredients suspended. The adjuvants can be chosen from amongthe thickeners, such as carboxymethylcellulose, polyvinyl pyrrolidone,gelatin and the alginates. Surfactants generally will serve to suspendthe active ingredients, particularly the fat-solublepropionate-enhancing compounds. Most useful for making suspensions inliquid nonsolvents are alkylphenol polyethylene oxide adducts,naphthalenesulfonates, alkylbenzene-sulfonates, and the polyoxyethylenesorbitan esters. In addition many substances, which affect thehydrophilicity, density and surface tension of the liquid, can assist inmaking suspensions in individual cases. For example, siliconeanti-foams, glycols, sorbitol, and sugars can be useful suspendingagents.

Additionally the subject compositions of this invention may beseparately administered, for example, by adding one directly to feedstuffs and co-administering the second material as a bolus tablet,drench, or capsule. Or each may be separately prepared and separatelyadded to feed stuffs in appropriate quantities and at appropriate times.For example, such a material as choline stearate, a fatty acid complexwhich may be used in the practice of this invention, may not beappropriate for incorporation into feed premixes because of its physicalcharacteristics. In such an instance the choline stearate compositioncould be provided separately in a suitable diluent such as, for example,corn flour, ground corn cob, hominy, corn glutenmeal, wheat middlings,soybean meal, soybean mill feed, rice mill by-product, and the like andmixtures thereof. A description of such suitable diluents may be foundin U.S. Pat. No. 4,394,377.

The Bacteria may be administered to an animal in a composition, apremix, that is then mixed into the animal feed supply. Such acomposition may comprise The Bacteria alone or The Bacteria may be mixedwith a carrier and/or with other drugs, vitamins, minerals, proteinconcentrates and similar feed supplements. These compositions may beprepared in dry granular powder form, as pellets, in the form of pastes,encapsulated to be rumen protected, or may be formulated as liquid feedsupplements and the like. Any type of feed may be medicated with suchcompositions, including common dry feed, liquid feeds, and pelletedfeeds. The methods of formulating supplemental materials into animalfeeds are well known. It is necessary only to calculate the amount ofeach compound, which it is desired to administer to each animal, to takeinto account the amount of feed per day that the animal eats and thenmix in the appropriate amount of The Bacteria. See U.S. Pat. No.4,394,377.

The compositions of the invention may be used as a feed additive premix,feed additive concentrate or feed additive supplement in which theactive ingredients are distributed uniformly throughout a standardorganic or inorganic animal feed carrier in a concentrated form which isconveniently packaged and shipped to the feed mixer. The grower or thefeed mixer then in turn mixes this premix, concentrate or supplementuniformly with a normal diet for the animal as desired. Examples ofcarriers for premix compositions are soybean meal, corn oil, groundcorn, barley, wheat, mineral mixtures containing, e.g., vermiculite ordiatomaceous earth, corn gluten meal, soy flour or other modestly pricededible ingredients.

The Bacteria may also be admixed with a suitable carrier such as anedible feed or feed component in the form of a feed additive supplement.Examples of such edible feed components are feed fortifiers andenhancers for preruminant bovine calves of any of the kinds disclosed inU.S. Pat. No. 6,156,333. If to be fed free choice or as a supplement,The Bacteria is provided according to the anticipated daily consumptionof the supplement to provide a daily dose of each of these ingredientsin one of the ranges specified.

In addition, The Bacteria may be incorporated directly into feeds by amill or other feed supplier to provide a finished feed product to thegrower. A finished feed product could be made up of any of the variousgrains, lucerne, grasses, minerals, vitamins, protein supplements, drugsand the like which go into the formulation of a nutritionally completeruminant feed. The Bacteria may be mixed directly with cattle feed madeup of various components such as hay, straw, silage, cornstalks,cottonseed hulls, grain, oats, barley and cereal brans, particularly forthe ruminants; antioxidants, minerals, vitamins, anthelmintics, andother appropriate medicaments. See U.S. Pat. No. 4,394,377.Alternatively, The Bacteria may be incorporated into a liquid feed forpreruminant bovine calves of any of the kinds disclosed in U.S. Pat. No.6,156,333.

The Bacteria may be mixed into a suitable animal feed by any methodappropriate for mixing a bacterium into animal feed. Examples of suchmethods include but are not limited to the following: spraying TheBacteria onto dry feed and mechanically mixing The Bacteria into dry orliquid feed; top dress grain or concentrate mix.

The Bacteria of the present invention are also useful in treatingmedical conditions in humans that result from various mycobacterialinfections. These include Crohn's, leprosy, tuberculosis, Sarcoidosis,and diarrhea in immuno-compromised (AIDS) patents. Administration of TheBacteria or active fragments, proteins, secretions, etc. thereof byappropriate means known in the art to a human patient should demonstratea reduction of the symptoms of the disease or syndrome caused by themycobacterium.

Dietzia sp. 79793-74, ATCC Accession Number PTA4125 was identified as aunique bacterial contaminant during culturing of feces from MpTsero-positive and negative cows. The bacterial contaminant wassubsequently isolated, cultured, and identified as being of the Dietziagenus. On further investigation, Dietzia sp. 79793-74 was found tocompletely inhibit the growth of MpT when co-cultured with MpT. Thismethod of utilizing a sample isolated from an appropriate animal hostmay be used to isolate other bacteria, in addition to those of theDietzia genus, that inhibit the growth of MpT and that are thereforeuseful for treating diseases caused by MpT.

Other micro-organisms that inhibit the growth of various mycobacteriamay be isolated by similar means. Other mycobacteria that may beinhibited include M. tuberculosis, M. leprae, M. avium-intracellulare,M. bovis, M. cheloiei (also known as borstelense and abscessus), M.africanum, M. marinium (also known as balnei and platypoecilus, thecausative agent of “swimming pool granuloma”), M. buruli (also known asulcerans), M. fortuitum (also known as giae, minetti, and ranae), M.haemophilum, M. intracellulare, M. kansasii (also known as luciflavum),M. littorale (also known as xenopi), M. malmoense, M. marianum (alsoknown as scrofulaceum and paraffinicum), M. sinuae, M. szulgai, and M.ulcerans (which is the agent responsible for Buruli ulcer), M. avium(also known as brunense), M. flavascens, M. lepraemurium, and M.microti.

Bacteria of the Dietzia genus may be cultured by growing the bacteria onagar slants in Tryptose soy broth at 35° C. Large-scale production ofDietzia genus bacteria is achieved by growth in suspension in largebiofermenters. Viability of such bacteria is unchanged by storing at−20° C. In addition, bacteria of the Dietzia genus can be stored as afreeze-dried material at room temperature. Other storage methods thatmaintain bacterial viability that are known in the art may be used.

Active fragments of bacteria from the Dietzia genus and active moleculesisolated from such bacteria may be prepared by any known method forpreparing/identifying active fragments of bacteria and proteins secretedfrom bacteria. Such methods include but are not limited to thefollowing: sonication, osmotic shock, detergent lysis, high pressure,transfer appropriate DNA to other organisms, such as bacteria, plant oranimal that is then used as a feed additive as described previously.

The compositions and methods of the invention create several importantbenefits: an effective treatment for Johne's disease is provided whichwill increase profitability for farmers; products such as milk and meatwill be safer for human consumption by eliminating potential MpTcontamination; and a potentially effective non-antibiotic treatment forCrohn's patients as well as other diseases caused by mycobacteria, suchas tuberculosis and leprosy. The methodology also teaches a way toidentify bacteria that inhibit the growth of various mycobacteria.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific embodiments described herein. Such equivalents are within thescope of this invention.

EXAMPLES

The invention is further described in the following, non-limitingexamples.

Example 1 Antibodies in sera and Colostrum/Milk from Dietzia-SensitizedCows Cross-React with MpT as Determined in an Enzyme-LinkedImmunosorbent Assay (ELISA) Assay

Healthy cows that tested negative for MpT infection were injected withthe indicated doses of Dietzia sp. C79793-74, Accession No. PTA-4125.The titers of antibodies that are cross-reactive with MpT in milk andsera taken from the injected cows were determined by a standard ELISAassay that those skilled in the art will recognize or be able toascertain.

TABLE 1 Cow Injection ELISA Titers ID # Date Dose Date Milk Serum 51None Jul. 21, 1994 − ND Feb. 03, 1995 2 × 10¹⁰ IM Feb. 03, 1995 ND −Feb. 17, 1995 6 × 10¹⁰ IM Feb. 17, 1995 ND ND Mar. 14, 1995 4 × 10⁹ IMMar. 14, 1995 + 4.6 Apr. 04, 1995 4 × 10⁹ IM May 09, 1995 ++ ND Jul. 28,1995 + ND Mar. 08, 1996 ND 3.2 B-5 None Jul. 21, 1994 − ND Feb. 03, 19952 × 10¹⁰ IM Feb. 03, 1995 ND − Mar. 14, 1995 ND 2.3 Apr. 04, 1995 4 ×10⁹ IM May 09, 1995 − ND Jul. 27, 1995 + ND Mar. 08, 1996 ND − B-10 NoneJul. 17, 1994 − ND Feb. 03, 1995 ND − Feb. 17, 1995 4 × 10¹⁰ IM Mar. 14,1995 ND 2.5 Mar. 14, 1995 4 × 10⁹ IM May 09, 1995 − ND Apr. 04, 1995 4 ×10⁹ IM Mar. 08, 1996 ND 2.1 Key: − means negative; + means positive; NDmeans not done; IM means intramuscularly

The results in Table 1 show that cows immunized with Dietzia bacteriaATCC Accession No. PTA4125 produce antibodies that cross-react with MpT.Moreover, these antibodies are found in both serum and milk. The resultsare important because they provide a possible explanation for themechanism of action of the Dietzia bacteria: phagocytes infected withMpT can more readily take up Dietzia bacteria coated with antibodiesthat cross-react with MpT. Thus, the cross-reactive antibodies may serveto deliver Dietzia bacteria to cells infected with MpT.

Example 2 Treatment of Cows that are Sero-Positive for Johne's Disease

Cows afflicted with Johne's disease were treated according to the methodof the invention by administration of Dietzia bacteria ATCC AccessionNo. PTA4125. The clinical results with seven such cows are shown inTable 2 below. Each cow was administered a single oral dose of 2×10¹¹cfu, on a daily basis, in a single dose, of Dietzia sp. C79793-74, ATCCAccession No. PTA4125.

TABLE 2 Cow Calving Treatment ID # Dates Length Treatment ObservationsH-83 Aug. 27, 1997 9 months Began showing clinical Apr. 20, 1999symptoms on Jan. 1, 1998. Jul. 20, 2000 Began daily treatments Aug. 8,2001 Feb. 25, 1998. Recovered on Apr. 1, 1998. Had a relapse on Oct. 18,1998 and recovered by Oct. 23, 1998. Stopped treatment on Nov. 13, 1998.She has not had any further re- lapses but did have a borderline serumELISA on Oct. 15, 1999 which returned to negative on Jun. 28, 2000 andAug. 20, 2001. Fecal samples taken Nov. 29, 1999, Jun. 28, 2000, Jan. 1,2001 and Aug. 20, 2001 were negative. H-51 Nov. 5, 1996 3 months Signsof disease on Jul. 17, 1998 Aug. 1, 1998. Started treatment on Aug. 31,1998. Recovered. No further symptoms. H-81 Aug. 30, 1997 6 months Brokeon May 24, 1998; Mar. 26, 1999 started treatment on May 25, 1998.Recovered. No further symptoms. H-58 Nov. 3, 1996 10 months  Signs ofdisease on Jan. 10, 1998. Started treatment on Jan. 25, 1998. Recovered.No further symptoms. H-62 Oct. 25, 1996 10 months  Signs of disease onFeb. 1, 1998 Jan. 20, 1998. Started on treatment on Jan. 20, 1998.Recovered. Relapse on Oct. 29, 1998. Recovered. No further symptoms.H-53 Nov. 3, 1996 5 months Signs of disease on Sep. 4, 1998 Apr. 7,1998; Started treatment same day. Recovered. Moved to maternity pen onAug. 26, 1998. She quit eating on Sep. 1, 1998, calved with difficultyand died with no clinical signs. H-64 Oct. 21, 1996 7 months Minor signsof disease Mar. 26, 1998 in first lactation. Relapse during 2ndlactation on Apr. 7, 1998. Started treatment on Apr. 7, 1998. Althoughthere were times when she appeared to recover, we were not able to turnher around. She eventually died on Nov. 5, 1998 with severe diarrhea andwas quite emaciated.

The results in Table 2 show that treatment of MpT sero-positive animalswith Dietzia sp. C79793-74, ATCC Accession No. PTA-4125 is effective(and for H-83, curative). For instance, cow H-83, who was treated withDietzia sp. C79793-74, ATCC Accession No. PTA4125 for 9 months remainsin the herd as of May 01, 2002, ELISA negative, culture negative andclinically free, 38 months after treatment was terminated. Results alsosuggest that the best candidates for treatment are those withearly-stage disease. Any results such as those shown in Table 2, orsimilar thereto depending on the mammal to be treated is said to meetthe definition of “reducing or preventing the symptoms of a disease orsyndrome” as discussed in this application.

To summarize, a clinical study was undertaken in which adult dairycattle that displayed symptoms of Johne's disease were administeredDietzia sp. C79793-74, ATCC Accession No. PTA4125. In this study, thebacteria was given daily for 3-10 months. Of seven clinically sickanimals treated, six recovered. Moreover, four treated animals that wereMpT-sero-positive, but clinically asymptomatic, never developed disease.In contrast, the ten MpT-clinically sick animals that were not treatedwent on to succumb with overt clinical disease.

Example 3 Safety of BC: Administration of Dietzia to Calves and Mice

In addition to the cows that were administered live or killed Dietziasp. C79793-74, ATCC Accession No. PTA-4125, a number of calves and micewere also administered the same live bacteria. Two types of mice wereused. Eight immunologically competent, 2-3 month old A/J males and eightfemales (two of each per cage) were injected IP with 10⁸ cfu liveDietzia sp. C79793-74, ATCC Accession No. PTA-4125. They were thenmonitored for ten months for any signs of disease (weight loss,diarrhea) and reproductive problems. All activities monitored wereindistinguishable from those of untreated (control) animals. Inaddition, 10⁸ cfu live Dietzia sp. C79793-74, ATCC Accession No. PTA4125were injected intraperitoneally (IP) into mouse CB.17severe-combined-immunodeficient mice (SCID) mice. Just as was found withconventional mice, the injected bacteria manifested no detrimental overtreaction even though these mice, genetically, are immunologicallyincompetent (they lack functional T and B cells).

Seven bull calves destined to become steers were used to determine thesafety of 10⁹ cfu Dietzia sp. C79793-74, ATCC Accession No. PTA-4125given orally, as a single dose, on a daily basis, from birth up to 15days. Two bull calves served as controls and did not receive any of thebacteria. All animals were castrated and raised for slaughter. Duringtheir lives of 15-18 months, no signs of disease or sickness wereobvious. They were all similar in weight at slaughter. At approximatelyfour months of age, a sample of blood was taken from all nine calvesfrom which the prepared sera was tested for antibodies to MpT in anELISA assay. All animals were found to be negative, except one calf thathad a positive ELISA reading. This was expected since this calf receivedcolostrum from a Dietzia sp. C79793-74 hyper-immunized cow (H-51 inTable 1) possessing antibodies in her milk.

The above results show that live Dietzia sp. C79793-74 are notpathogenic when administered orally or injected.

Example 4 Treatment of Clinically Sick and/or Asymptomatic Cows

A large study was initiated in which liquid or freeze-dried Dietzia sp.C79793-74 is given to clinically sick and/or asymptomatic cows that areeither

a) Sero-ELISA positive, Sero-AGID negative, fecal negative

b) Sero-ELISA positive, Sero-AGID positive, fecal negative

c) Sero-ELISA positive, Sero-AGID negative, fecal positive

d) Sero-ELISA positive, Sero-AGID positive, fecal positive

e) Sero-ELISA negative, Sero-AGID negative, fecal positive.

After 7 months, the preliminary results indicate that the dose of thebacteria necessary to prevent death of Johne's positive cows isdependent upon the body weight of the animal being treated. Holsteinsfor example that weigh up to 2× more than Jerseys, need at least 2×higher dose of bacteria.

Animals that were ELISA positive and agar gel immunodiffusion (AGID)positive were more difficult to maintain alive than those that were onlyELISA positive. Survival did not appear to depend upon whether an animalshed or did not shed MpT.

Of 19 animals (out of 36) found to be shedding MpT in feces, after 6weeks of treatment, only one was found to still be shedding. Of 8animals that calved while on treatment, none had detectable MpT in theircolostrums. This is contrary to what is published. These results suggestthat is it possible that the treatment prevented shedding in colostrums.

A number of animals (12) died during treatment, most due tocomplications such as displaced abomasums. Only 4 died due to Johne'sand they were all AGID positive when started on treatment.

In tissues of two animals that were euthanized and autopsied, no Dietziawere detected.

Weight changes post treatment of two animals that were or becameclinically very sick are shown in Table 3 below.

TABLE 3 weights on Cow Sep. 1, Oct. 24, Dec. 06, Jan. 2, Feb. 10, # 20012001 2001 2002 2002 G-4 945 1095 1160 1190 1090 R100 1000 840 940 1045 1030* Weight prior to treatment *calved

Treatment appears to reduce shedding of MpT in both feces and milk. Verysick animals gain weight. No Dietzia was found systemically in autopsiedcows.

Example 5 Long Term Treatment of Antibody Positive Cows

Three cows that tested sero-positive for Johne's disease were treatedaccording to the method of the present invention by administration ofDietzia bacteria ATCC Accession No. PTA4125. Each cow was administered asingle oral dose of 10¹² cfu, on a daily basis of Dietzia sp. C79793-74,ATCC Accession No. PTA-4125. The serum antibody titers specific for MpTas determined in an ELISA assay for these three cows was measuredperiodically during the treatment period. The results of thesemeasurements are presented in Table 4 below. A value of zero indicatesthat a test serum is void of antibodies for MpT, whereas a value greaterthan 1 indicates that the sera is from an animal that is/was infectedwith MpT.

TABLE 4 Cow Antibody Level in Serum On Day # Start Date 18 70 95 147 183235 273 335 341 400 425 490 525 590 13 2.2 1.7 1.7 1.6 0 1.8 0 198 2.62.9 2.4 1.8 0 2.0 0 0 1734 2.2 1.9 3.1 2.2 1.8 2.0 2.0 0

Cow 13 died from birthing complications and was autopsied. The autopsyshowed no signs of Johne's disease and no growth of MpT from the cow stissue. The immune systems of the cows were healthy following treatment,therefore, the disappearance of antibodies in the sera of the cowssuggest that MpT was no longer present in the animals.

1. A composition, comprising a pharmacologically active dose of anisolated bacterium of the genus Dietzia, wherein said isolated bacteriumis deposited with the American Type Culture Collection as AccessionNumber PTA-4125.
 2. The composition of claim 1 further comprising ananimal feed.
 3. The composition of claim 2 where the animal feed isselected from the group consisting of grain, hay, straw, silage,cornstalks, corn, corn meal, corn glutenmeal, soybean meal, soy flour,soybean mill feed, cottonseed hulls, oats, barley, hominy, cereal brans,rice mill by-product and mixtures thereof.
 4. The composition of claim 1further comprising a feed additive.
 5. A composition comprising a dosein the range of 10⁹ to 10¹⁴ cfu of an isolated bacterium of the genusDietzia deposited with the American Type Culture Collection as AccessionNumber PTA-4125 such that the composition is capable of reducing orpreventing the symptoms of a disease or syndrome whose causative agentis Mycobacterium paratuberculosis.